CN114728875A

CN114728875A - Metal salts and their use

Info

Publication number: CN114728875A
Application number: CN202080006947.7A
Authority: CN
Inventors: S·瓦尔德; E·J·马丁内斯; S·斯特拉特福德; A·比伊斯特; J·本森; J·拉夫瑞
Original assignee: Insbona
Current assignee: Insbona
Priority date: 2019-12-13
Filing date: 2020-12-11
Publication date: 2022-07-08
Also published as: US11878956B2; US20230095288A1; MX2022007164A; TW202136188A; US20210179539A1; AU2020401286A1; US11459292B2; US11174220B2; JP2023509845A; EP4073025B1; US20220041541A1; EP4073025A4; WO2021119397A9; ZA202206588B; CA3161274A1; WO2021119397A1; EP4073025A1

Abstract

The present invention relates to 2- [3- [ (3) exhibiting improved physical properties and stabilityR) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]A metal salt of acetic acid. The invention also relates to pharmaceutical compositions comprising an effective amount of a metal salt, and methods of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition comprising a salt of the invention.

Description

Metal salts and their use

Background

The Liver X Receptor (LXR) is a nuclear receptor transcription factor. LXR modulators have been found to be useful in the treatment of a variety of diseases, including cancer. There is a need to provide salts of such compounds with improved stability and physical properties.

Summary of The Invention

The present invention provides metal salts of LXR β agonists. The invention also provides methods of making such metal salts of LXR β agonists, pharmaceutical compositions comprising said metal salts, and methods of treating cancer using such compositions.

Thus, in one aspect, the invention features 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]A metal salt (e.g., a pharmaceutically acceptable metal salt) of acetic acid (compound 1).

In certain embodiments, the 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]The metal salt of acetic acid is a polyvalent metal salt.

In certain embodiments, the 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy group]Phenyl radical]The metal salt of acetic acid is a zinc salt, for example, the 2:1 (compound 1: zinc) salt.

In certain embodiments, the 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]The metal salt of acetic acid is an aluminum salt, for example, a 3:1 (compound 1: aluminum) salt.

In certain embodiments, the 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]The metal salt of acetic acid is a bismuth salt, for example, a 3:1 (compound 1: bismuth) salt.

In certain embodiments, the 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]The metal salt of acetic acid is a calcium salt, for example, the 2:1 (compound 1: calcium) salt.

In certain embodiments, the metal salt is amorphous. In certain embodiments, the metal salt (e.g., amorphous zinc salt) has a peak area of about 1590 ± 10 cm as measured by fourier transform infrared spectroscopy (FTIR)^-1A peak at increased intensity relative to the free acid and at about 1710. + -.10 cm^-1A peak with reduced intensity relative to the free acid. In certain embodiments, the metal salt has a mass loss due to decomposition of less than 1% as measured by thermogravimetric analysis.

In another aspect, the invention features a method of producing a zinc salt of compound 1 (e.g., a 2:1 zinc salt). The method comprises combining compound 1 or a salt thereof (e.g., a 1:1 sodium salt or a free compound) and a zinc salt (e.g., zinc chloride or zinc acetate) in amounts sufficient to produce a zinc salt of compound 1.

In certain embodiments of the method of producing a zinc salt, the method comprises dissolving compound 1 or a salt thereof and a zinc salt in a solvent to form a mixture. In certain embodiments, the solvent is water. In certain embodiments, the solvent is a mixture of an organic solvent (e.g., methanol) and water (e.g., 9:1 organic solvent and water by volume). In certain embodiments of the method of producing a zinc salt, the method further comprises cycling the temperature of the mixture between ambient temperature and 40 ℃. In certain embodiments of the method of producing a zinc salt, wherein the cycling is performed for 24 hours.

In another aspect, the invention features a pharmaceutically acceptable zinc salt of compound 1 produced by any one of the aforementioned methods.

In another aspect, the invention features a method of producing an aluminum salt of compound 1 (e.g., a 3:1 aluminum salt). The method comprises combining compound 1 or a salt thereof (e.g., a 1:1 sodium salt or a free compound) and an aluminum salt (e.g., aluminum sulfate) in amounts sufficient to produce an aluminum salt of compound 1.

In another aspect, the invention features a pharmaceutical composition containing any of the foregoing metal salts and a pharmaceutically acceptable excipient.

In certain embodiments, the pharmaceutical composition comprises less than 1.5% by weight sodium. In certain embodiments, the pharmaceutical composition is substantially free of the 1:1 sodium salt of compound 1. In certain embodiments, the pharmaceutical composition is in a unit dosage form.

In another aspect, the invention features a method of treating cancer. The method comprises administering an effective amount of any of the foregoing salts or pharmaceutical compositions.

In certain embodiments of the methods of treating cancer, the subject has a cancer that has failed to respond to a previously administered immunotherapy (e.g., the subject's cancer has progressed despite treatment with immunotherapy).

In certain embodiments of the methods of treating cancer, the cancer is resistant to immunotherapy (e.g., the cancer has been determined to be resistant to immunotherapy, such as by genetic markers or levels of MDSCs (e.g., monocyte and/or granulocyte MDSCs) in a sample, or may be resistant to immunotherapy, such as a cancer that has failed to respond to immunotherapy).

In another aspect, the invention features a method of treating a cancer that has failed to respond to immunotherapy in a subject. The method comprises administering to the subject an effective amount of any of the foregoing salts or pharmaceutical compositions in combination with immunotherapy.

In another aspect, the invention features a method of treating a cancer resistant to immunotherapy in a subject. The method comprises administering to the subject an effective amount of any of the foregoing salts or pharmaceutical compositions in combination with immunotherapy.

In certain embodiments of any of the methods of treating cancer, the cancer is breast cancer, colon cancer, renal cell carcinoma, lung cancer (e.g., non-small cell lung cancer), hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, glioblastoma, diffuse large B-cell lymphoma, leukemia, or melanoma. In certain embodiments, the cancer is a metastatic cancer. In certain embodiments of any of the methods of treating cancer, the effective amount is an amount effective to inhibit metastatic colonization of the cancer.

In certain embodiments of any of the methods of treating cancer, the cancer is a drug-resistant cancer or a cancer that has failed response to a previous therapy (e.g., a cancer that is resistant to a previous therapy, or a cancer that has failed response to a previous therapy using vemurafenib, dacarbazine, a CTLA-4 inhibitor, a PD-1 inhibitor, an interferon therapy, a BRAF inhibitor, a MEK inhibitor, radiation therapy, temozolomide, irinotecan, CAR-T therapy, herceptin, perjeta, tamoxifen, hiroda, docetaxel, a platinum agent such as carboplatin, a taxane such as paclitaxel and docetaxel, an ALK inhibitor, a MET inhibitor, clofibrate, injectable paclitaxel, doxorubicin, gemcitabine, avastin, halaven, neratinib, a PARP inhibitor, brilanstrant, an mTOR inhibitor, topotecan, a nosable, a, VEGFR2 inhibitors, folate receptor antagonists, democizumab, fotambulin, or PDL-1 inhibitors).

In one embodiment of any of the methods of treating cancer, the immunotherapy, when present, is a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, or an adoptive T-cell transfer therapy. In certain embodiments, the immunotherapy comprises a PD-1 inhibitor such as a PD-1 antibody, a PD-L1 inhibitor such as a PD-L1 antibody, a CTLA-4 inhibitor such as a CTLA-4 antibody, a CSF-1R inhibitor, an IDO inhibitor, a1 adenosine inhibitor, A2A adenosine inhibitor, A2B adenosine inhibitor, A3A adenosine inhibitor, an arginase inhibitor, or an HDAC inhibitor. In certain embodiments, the immunotherapy comprises a PD-1 inhibitor (e.g., nivolumab, pembrolizumab, pidilizumab, BMS 936559, and astuzumab). In certain embodiments, the immunotherapy comprises a PD-L1 inhibitor (e.g., atelizumab and dulvacizumab). In certain embodiments, the immunotherapy comprises a CTLA-4 inhibitor (e.g., ipilimumab). In certain embodiments, theImmunotherapy includes CSF-1R inhibitors (e.g., piroxicam and 4- (2, 4-difluoroanilino) -7-ethoxy-6- (4-methylpiperazin-1-yl) quinoline-3-carboxamide). In certain embodiments, the immunotherapy comprises an IDO inhibitor (e.g., norharpagne, rosmarinic acid, and alpha-methyl-tryptophan). In certain embodiments, the immunotherapy comprises an A1 adenosine inhibitor (e.g., 8-cyclopentyl-1, 3-dimethylxanthine, 8-cyclopentyl-1, 3-dipropylxanthine, 8-phenyl-1, 3-dipropylxanthine, paminotheophylline, BG-9719, tonapofylline, FK-453, FK-838, rolifylline, or N-0861). In certain embodiments, the immunotherapy comprises an A2A adenosine inhibitor (e.g., ATL-4444, istradefylline, MSX-3, propranolol, SCH-58261, SCH-412348, SCH-442416, 2-butyl-9-methyl-8- (triazol-2-yl) purin-6-amine, VER-6623, VER-6947, VER-7835, viadenant, or ZM-241,385). In certain embodiments, the immunotherapy comprises an A2B adenosine inhibitor (e.g.,N- [5- (1-cyclopropyl-2, 6-dioxo-3-propyl-7)H-purin-8-yl) pyridin-2-yl]-N-ethylpyridine-3-carboxamide, 3-ethyl-1-propyl-8- [1- [ [3- (trifluoromethyl) phenyl ] methyl ester]Methyl radical]Pyrazol-4-yl]-7H-purine-2, 6-dione, MRS-1706, MRS-1754,N- [2- [ [ 2-phenyl-6- [4- (3-phenylpropyl) piperazine-1-carbonyl]-7H-pyrrolo [2,3-d]Pyrimidin-4-yl]Amino group]Ethyl radical]Acetamide, PSB-603, PSB-0788 or PSB-1115). In certain embodiments, the immunotherapy comprises an A3A adenosine inhibitor (e.g., KF-26777, MRS-545, MRS-1191, MRS-1220, MRS-1334, 6-ethyl-5-ethylsulfanylcarbonyl-2-phenyl-4-propylpyridine-3-carboxylic acid propyl ester, MRS-3777, MRE-3005-F20, MRE-3008-F20, PSB-11, OT-7999, VUF-5574, and SSR 161421). In certain embodiments, the immunotherapy comprises an arginase inhibitor (e.g., an arginase antibody, (2S) - (+) -amino-5-iodoacetaminopentanoic acid, NG-hydroxy-L-arginine, (2S) - (+) -amino-6-iodoacetamidohexanoic acid, or (R) -2-amino-6-borono-2- (2- (piperidin-1-yl) ethyl) hexanoic acid.

In another embodiment of any of the methods of treating cancer, the method further comprises administering to the subject an additional anti-cancer therapy (e.g., an antiproliferative agent).

In certain embodiments, the antiproliferative agent is: chemotherapeutic or cytotoxic agents, differentiation inducing agents (e.g., retinoic acid, vitamin D, cytokines), hormonal agents, immunological agents, or anti-angiogenic agents. Chemotherapeutic and cytotoxic agents include, but are not limited to: alkylating agents, cytotoxic antibiotics, antimetabolites, vinca alkaloids, etoposide and others (e.g., paclitaxel, taxol, docetaxel, taxotere, cisplatin). A list of additional compounds with antiproliferative activity can be found in: l, Brunton, B, Chabner and B, Knollman (eds.). Goodman and Gilman's The pharmaceutical Basis of Therapeutics, twelfth edition, 2011, McGraw Hill company, New York, NY.

In certain embodiments, the antiproliferative agent is a PD-1 inhibitor, a VEGF inhibitor, a VEGFR2 inhibitor, a PD-L1 inhibitor, a BRAF inhibitor, a CTLA-4 inhibitor, a MEK inhibitor, an ERK inhibitor, vemurafenib, dacarbazine, trimetinib, dabigatran, dutvacizumab, an mTOR inhibitor, CAR-T therapy, abiraterone, enzalutamide, apalutamide, 5-fluorouracil (5-FU), FOLFOX (i.e., folinic acid, 5-fluorouracil and oxaliplatin), FOLFIRI (i.e., folinic acid, 5-fluorouracil and irinotecan), herceptin, hiloda, PD-1 antibodies (e.g., pembrolizumab or nivolumab), PD-L1 antibodies, CTLA-4 antibodies (e.g., ipilimumab), ramucirumab, ridapepimot, glembatumumab, vedotin, ANG1005 and/or ANG 4043.

In certain embodiments of any of the methods of treating cancer, the cancer is renal cell carcinoma and the antiproliferative agent is a PD-1 inhibitor, a PD-L1 inhibitor, or an mTOR inhibitor. In other embodiments, the cancer is diffuse large B-cell lymphoma and the antiproliferative agent is CAR-T therapy. In certain embodiments, the cancer is prostate cancer and the antiproliferative agent is abiraterone, enzalutamide, or apaluramide. In certain embodiments, the cancer is hepatocellular carcinoma, gastric cancer, or esophageal cancer and the antiproliferative agent is 5-FU, FOLFOX, FOLFIRI, herceptin, or hiloda. In certain embodiments, the cancer is a sarcoma and the antiproliferative agent is gemcitabine. In other embodiments, the cancer is pancreatic cancer and the antiproliferative agent is irinotecan, cisplatin, paclitaxel for injection, a taxane (e.g., paclitaxel or docetaxel), or capecitabine.

The method of treating cancer may further comprise administering antiproliferative agents such as alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyl transferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF α agonists/antagonists, endothelin a receptor antagonists, retinoic acid receptor agonists, immune-modulators, hormonal and anti-hormonal agents, photodynamic agents, tyrosine kinase inhibitors, antisense compounds, corticosteroids, HSP90 inhibitors, proteosome inhibitors (e.g., marizomib), CD40 inhibitors, anti-CSI antibodies, FGFR3 inhibitors, VEGF inhibitors, MEK inhibitors, cyclin D1 inhibitors, NF-kB inhibitors, anthracyclines, histone deacetylases, kinesin inhibitors, phosphatase inhibitors, COX2 inhibitors, mTOR inhibitors, calcineurin antagonists, IMiD, and/or other agents useful for treating proliferative diseases.

In certain embodiments of any of the methods of treating cancer, the cancer is breast cancer such as triple negative breast cancer, colon cancer, renal cell carcinoma, lung cancer (e.g., non-small cell lung cancer), hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, glioblastoma, diffuse large B-cell lymphoma, leukemia (e.g., acute myeloid leukemia), or melanoma. In certain embodiments of any of the methods of treating cancer, the cancer is melanoma. In certain embodiments of any of the methods of treating cancer, the cancer is breast cancer. In certain embodiments of any of the foregoing methods, the cancer is renal cell carcinoma. In certain embodiments of any of the methods of treating cancer, the cancer is pancreatic cancer. In certain embodiments of any of the methods of treating cancer, the cancer is non-small cell lung cancer. In certain embodiments of any of the methods of treating cancer, the cancer is colon cancer. In certain embodiments of any of the methods of treating cancer, the cancer is ovarian cancer. In certain embodiments of any of the methods of treating cancer, the cancer is glioblastoma. In certain embodiments of any of the methods of treating cancer, the cancer is prostate cancer. In certain embodiments of any of the methods of treating cancer, the cancer is diffuse large B-cell lymphoma. In certain embodiments, the cancer is leukemia (e.g., acute myeloid leukemia).

In a particular embodiment of any method of treating cancer, the cancer is melanoma (e.g., metastatic melanoma) that is resistant to or has failed a response to a previous treatment with vemurafenib, dacarbazine, interferon therapy, a CTLA-4 inhibitor, a BRAF inhibitor, a MEK inhibitor, a PD1 inhibitor, a PDL-1 inhibitor, and/or CAR-T therapy. In certain embodiments of any of the methods of treating cancer, the cancer is glioblastoma that is resistant to or has failed to respond to previous treatments with temozolomide, radiation therapy, avastin, irinotecan, a VEGFR2 inhibitor, CAR-T therapy, and/or an mTOR inhibitor. In certain embodiments of any of the methods of treating cancer, the cancer is a non-small cell lung cancer such as metastatic non-small cell lung cancer (e.g., EGFR-wild type non-small cell lung cancer and/or squamous non-small cell lung cancer) that is resistant to or has failed a previous treatment with an EGFR inhibitor, a platinum agent (e.g., carboplatin), avastin, an ALK inhibitor, a MET inhibitor, a taxane (e.g., paclitaxel and/or docetaxel), a robusta, petasite, radiation therapy, a PD-1 inhibitor, a PD-L1 inhibitor, and/or CAR-T therapy. In certain embodiments of any of the methods of treating cancer, the cancer is breast cancer that is resistant to or has failed a previous treatment with herceptin, perjeta, tamoxifen, hiloda, docetaxel, carboplatin, paclitaxel for injection, doxorubicin, gemcitabine, avastin, halaven, neratinib, PARP inhibitors, PD-1 inhibitors, PD-L1 inhibitors, CAR-T therapy, apaluran, and/or mTOR inhibitors (e.g., triple negative breast cancer). In certain embodiments of any of the methods of treating cancer, the cancer is an ovarian cancer (e.g., advanced ovarian cancer) that is resistant to or has failed a response to a previous treatment with a PARP inhibitor, avastin, a platinum agent such as carboplatin, paclitaxel, docetaxel, topotecan, a tonic, a VEGR2 inhibitor, a folate receptor antagonist, a PD-1 inhibitor, a PD-L1 inhibitor, CAR-T therapy, demcimab, and/or fotambulin.

In certain embodiments of any of the methods of treating cancer, the additional anti-cancer therapy, if present, comprises chemotherapy.

In certain embodiments of any of the methods of treating cancer, the chemotherapy comprises docetaxel. In certain embodiments, the method comprises administering to the subject an effective amount of docetaxel once every seven days. In certain embodiments, the effective amount of docetaxel is at least 28 mg/m². In certain embodiments, the effective amount of docetaxel is about 28 mg/m²To about 35 mg/m²。

In certain embodiments of any of the methods of treating cancer, the additional anti-cancer therapy comprises chemotherapy and immunotherapy. In certain embodiments of any of the methods of treating cancer, the anti-cancer therapy comprises carboplatin or cisplatin, pemetrexed, and pembrolizumab. In certain embodiments, the method comprises administering to the subject an effective amount of pembrolizumab 1 time every 21 days. In certain embodiments, the effective amount of pembrolizumab is about 200 mg. In certain embodiments, the method comprises administering to the subject an effective amount of carboplatin or cisplatin 1 time every 21 days. In certain embodiments, the effective amount of carboplatin or cisplatin is calculated using the formula: total dose (mg) = (target area under curve) x (glomerular filtration rate of subject +25), wherein the curveThe target area under the line was 4 mg/mL min to 6 mg/mL min, and glomerular filtration rate of the subjects was measured by Cr-EDTA clearance. In certain embodiments, the effective amount of carboplatin or cisplatin is about 300 mg/m²To about 360 mg/m². In certain embodiments, the method comprises administering to the subject an effective amount of pemetrexed 1 time every 21 days. In certain embodiments, the effective amount of pemetrexed is 500 mg/m². In certain embodiments of any of the methods of treating cancer, the method further comprises administering to the subject an effective amount of folic acid, vitamin B12, and/or a corticosteroid. In certain embodiments, the method comprises administering to the subject an effective amount of a corticosteroid 2 times a day for 3 days, followed by administration of pemetrexed.

In certain embodiments of any of the methods of treating cancer, the method further comprises administering to the subject an effective amount of a statin (e.g., rosuvastatin or atorvastatin).

In certain embodiments of any of the methods of treating cancer, the method further comprises administering to the subject an effective amount of an antiemetic agent (e.g., ondansetron, granisetron, palonosetron, metoclopramide, haloperidol, dexamethasone, aprepitant, fosaprepitant, lorazepam, dronabinol, prochlorperazine or chlorpromazine), an antidiarrheal agent (e.g., an opiate agonist or octreotide), an appetite stimulant (e.g., megestrol acetate, metoclopramide, dronabinol, prednisone or dexamethasone), a systemic stimulant, a bisphosphonate (e.g., etidronate, clodronate, tiludronate, pamidronate, neridronate, opadronate, alendronate, ibandronate, or zoledronate), a gonadotropin-releasing hormone agonist (e.g., brethrene, histrelin, or zoledronate), a gonadotropin-releasing hormone agonist (e.g., a combination of these compounds, Leuprolide, triptorelin, goserelin, or nafarelin) and/or a growth factor (e.g., filgrastim).

In certain embodiments of any of the methods of treating cancer, the cancer is resistant to an anticancer therapy (e.g., platinum-containing chemotherapy, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, antimitotic agents, topoisomerase inhibitors, antimetabolites, angiogenesis inhibitors, kinase inhibitors, and/or alkylating agents). In certain embodiments of any of the methods of treating cancer, the cancer progresses upon or after treatment with an anti-cancer therapy (e.g., platinum-containing chemotherapy, PD-1 inhibitors, PD-L1 inhibitors, angiogenesis inhibitors, kinase inhibitors, and/or alkylating agents). In certain embodiments of any of the methods of treating cancer, the cancer has been determined, or predicted to be, resistant to an anticancer therapy (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a topoisomerase inhibitor, an antimetabolite, an angiogenesis inhibitor, a kinase inhibitor, and/or an alkylating agent).

In certain embodiments of any of the methods of treating cancer, the cancer has a PD-L1 expression level of less than 1% when tested in an immunohistochemistry assay (e.g., an immunohistochemistry assay with a tumor proportion score). In certain embodiments of any of the methods of treating cancer, the cancer has a PD-L1 expression level of about 1% when tested in an immunohistochemistry assay (e.g., an immunohistochemistry assay with a tumor proportion score). In certain embodiments of any of the methods of treating cancer, the cancer has a PD-L1 expression level of about 1% to about 49% (e.g., about 1% to about 20%, about 5% to about 30%, about 15% to about 40%, about 25% to about 49%) when tested in an immunohistochemistry assay (e.g., an immunohistochemistry assay with a tumor proportion score). In certain embodiments of any of the methods of treating cancer, the cancer is metastatic and/or locally advanced. In certain embodiments of any of the methods of treating cancer, the cancer is unresectable.

Definition of

The term "administering" as used herein means administering a composition (e.g., a salt or a formulation comprising a salt as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any suitable route. For example, in certain embodiments, administration can be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and vitreous.

"biological sample" or "sample" refers to a fluid or solid sample from a subject. The biological sample may comprise cells; nucleic acid, protein or membrane extracts of cells; or blood or biological fluids including (e.g., plasma, serum, saliva, urine, bile). Solid biological samples include samples taken from stool, rectum, central nervous system, bone, breast tissue, kidney tissue, cervix, endometrium, head or neck, gall bladder, parotid gland tissue, prostate, brain, pituitary, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, and thymus. Fluid biological samples include samples taken from blood, serum, plasma, pancreatic fluid, CSF, semen, prostatic fluid, semen, urine, saliva, sputum, mucus, bone marrow, lymph, and tears. Samples can be obtained by standard methods including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a blood, plasma, or serum sample. In certain embodiments, the biological sample is a tumor sample from a biopsy.

The term "cancer" means any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas.

By "determining the level of a cell type" is meant detecting a cell type, either directly or indirectly, by methods known in the art. "directly determining" refers to processing (e.g., performing an assay or test on a sample, or "analyzing a sample" as the term is defined herein) to obtain a physical entity or value. "indirectly determining" refers to receiving a physical entity or value from another party or source (e.g., a third party laboratory that directly obtains the physical entity or value). Methods of measuring cellular levels typically include, but are not limited to, flow cytometry and immunohistochemistry. Exemplary methods are provided herein. In certain embodiments of any of the foregoing methods, the level of MDSCs and/or activated T-cells can be determined as described in Iclozan et al, Cancer immunol. 2013, 62(5): 909-918. In certain embodiments of any of the foregoing methods, the methods can be as described in Kitano et al Cancer immunol. res. 2014, 2 (8); 812 — 821 the level of MDSCs and/or activated T-cells was determined as described.

As used herein, a "cancer determined to be drug-resistant" refers to a cancer that is drug-resistant based on unresponsiveness or reduced responsiveness to a chemotherapeutic agent, or that is predicted to be drug-resistant based on a prognostic assay (e.g., a gene expression assay).

A "drug-resistant" cancer refers to a cancer that does not respond or exhibits a reduced response to one or more chemotherapeutic agents (e.g., any of the agents described herein).

The term "effective amount" refers to an amount sufficient to treat a disease, disorder and/or condition when administered to a population suffering from or susceptible to such a disease, disorder and/or condition according to a therapeutic dosing regimen. In certain embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity and/or delays the onset of one or more symptoms of a disease, disorder, and/or condition. One of ordinary skill in the art will appreciate that the term "effective amount" does not actually require that successful treatment be achieved in a particular individual. Conversely, an effective amount may be an amount that provides a particular desired pharmacological response in a significant number of subjects when administered to a patient in need of such treatment. It is specifically understood that a particular subject may actually be "in an effective amount" refractory. As just one example, refractory subjects may have low bioavailability, making clinical efficacy unavailable. In certain embodiments, reference to an effective amount can be a reference to an amount measured in one or more specific tissues (e.g., a tissue affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). One of ordinary skill in the art will appreciate that in certain embodiments, an effective amount may be formulated and/or administered in a single dose. In certain embodiments, an effective amount may be formulated and/or administered in multiple doses, e.g., as part of a dosing regimen.

The term "failure to respond to a prior therapy" or "refractory to a prior therapy" as used herein refers to a cancer that has progressed despite treatment with the therapy.

"level" refers to the level of a cell type compared to a reference. The reference may be any useful reference as defined herein. A "reduced level" or "increased level" of a cell type refers to a reduction or increase in the level of the cell as compared to a reference (e.g., a reduction or increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more; a reduction or increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100% or about 200%, a reduction or increase of less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold or less; or an increase of more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, or less than about 3-fold of a reference, About 4.5 times, about 5.0 times, about 10 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 100 times, about 1000 times, or more). The level of a cell type can be expressed in terms of mass/volume (e.g., g/dL, mg/mL, μ g/mL, ng/mL) or as a percentage of total cells in a sample. In certain embodiments of any one of the aforementioned methods, the reference is a sample from a healthy subject (such as a subject that does not have cancer). In certain embodiments of any of the foregoing methods, the reference is an artificial sample having a level (e.g., a level of MDSCs such as monocyte and/or granulocyte MDSCs or activated T-cells) that is demonstrated to be beneficial in the treatment of the disorder.

As used herein, a "metastatic nodule" refers to an aggregation of tumor cells in vivo at a site other than the site of the original tumor.

As used herein, "metastatic tumor" refers to a tumor or cancer in which tumor-forming cancer cells have a high potential or have begun to metastasize or spread from one location to another location or locations within the subject via the lymphatic system or via hematogenous spread, e.g., to create a secondary tumor within the subject. Such metastatic behaviour may be indicative of malignancy. In some cases, metastatic behavior may be associated with an increase in cell migration and/or invasion behavior of tumor cells.

Examples of cancers that may be defined as metastatic include, but are not limited to: lung cancer (e.g., non-small cell lung cancer), breast cancer, ovarian cancer, colorectal cancer, biliary tract cancer, bladder cancer, brain cancer (including glioblastoma and medulloblastoma), cervical cancer, choriocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, hematologic neoplasms, multiple myeloma, leukemia, intraepithelial neoplasms, liver cancer, lymphoma, neuroblastoma, oral cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer (including melanoma), basal cell carcinoma, squamous cell carcinoma, testicular cancer, stromal tumor, germ cell tumor, thyroid cancer, and renal cancer.

As used herein, "non-metastatic, cell-migratory cancer" refers to a cancer that migrates without passing through the lymphatic system or through blood borne transmission.

The term "pharmaceutical composition" as used herein means an active compound or a pharmaceutically acceptable salt thereof formulated with one or more pharmaceutically acceptable carriers. In certain embodiments, the active compound or salt is present in a unit dosage amount suitable for administration in a treatment regimen that demonstrates a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In certain embodiments, the pharmaceutical compositions may be specifically formulated for administration in solid or liquid form, including those suitable for use in: oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablets, e.g., those for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or a sustained release formulation; topical application, e.g., as a cream, ointment, or controlled release patch or spray for application to the skin, lung, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; lingually; through the eye; transdermally; or nasal, pulmonary and applied to other mucosal surfaces.

As used herein, "pharmaceutically acceptable excipient" means any inactive ingredient (e.g., a vehicle capable of suspending or dissolving an active compound) that has non-toxic and non-inflammatory properties in a subject. Typical excipients include, for example, antiadherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, flavorants, glidants (flow promoters), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or water of hydration.

The term "pharmaceutically acceptable salts" as used herein refers to those salts of the compounds described herein which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al,J. Pharmaceutical Sciences66:1-19, 1977 andHandbook of Pharmaceutical Salts: Properties, Selection, and Use(edit p.h. Stahl and c.g. Wermuth), Wiley-VCH, 2008. The salts may be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free basic groups with a suitable organic acid.

As used herein, "progression-free survival" refers to the length of time during and after medical treatment or therapy during which the disease being treated (e.g., cancer) does not become worse.

"proliferation" as used in this application relates to the regeneration or reproduction of similar forms (cells) caused by constituent (cellular) elements.

"slowing the propagation of metastasis" as used herein means reducing or stopping the formation of new sites; or reduce, stop or reverse tumor burden.

The term "subject" as used herein means a human or non-human animal (e.g., a mammal such as a non-human primate, horse, cow, or dog).

The term "substantially" refers to a qualitative condition that exhibits all or nearly all of a range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, go to completion and/or progress to completion or to achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

By "treatment regimen" is meant a quantitative administration regimen, the administration of which between related populations correlates with a desired or beneficial therapeutic outcome.

The term "treating" (also referred to as "treating") in its broadest sense refers to any administration of a substance (e.g., a provided composition) that partially or completely alleviates, ameliorates, alleviates, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In certain embodiments, such treatment may be administered to a subject who does not exhibit signs of the associated disease, disorder, and/or condition and/or who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, in certain embodiments, treatment may be administered to a subject exhibiting one or more defined signs of an associated disease, disorder, and/or condition. In certain embodiments, the treatment can pertain to a subject that has been diagnosed as having an associated disease, disorder, and/or condition. In certain embodiments, the treatment may be of a subject known to have one or more susceptibility factors statistically correlated with an increased risk of developing the associated disease, disorder, and/or condition.

The term "PD-1 inhibitor" as used herein denotes a compound, such as an antibody, which is capable of inhibiting the activity of a protein encoded by the PDCD1 gene in humans. Known PD-1 inhibitors include nivolumab, pembrolizumab, pidilizumab, BMS 936559, and astuzumab.

The term "PD-L1 inhibitor" as used herein denotes a compound, such as an antibody, which is capable of inhibiting the activity of the protein encoded by the CD274 gene in humans. Known PD-L1 inhibitors include atelizumab and doxab.

The term "CTLA-4 inhibitor" as used herein refers to a compound, such as an antibody, which is capable of inhibiting the activity of a protein encoded by the CTLA4 gene in humans. Known CTLA-4 inhibitors include ipilimumab.

The term "CSF-1R inhibitor" as used herein denotes a compound, such as an antibody, which is capable of inhibiting the activity of the protein encoded by the CSF1R gene in humans. Known CSF-1R inhibitors include piroxicam and 4- (2, 4-difluoroanilino) -7-ethoxy-6- (4-methylpiperazin-1-yl) quinoline-3-carboxamide.

The term "IDO inhibitor" as used herein means a compound, such as an antibody, that is capable of inhibiting the activity of the protein encoded by the IDO1 gene in humans. Known IDO inhibitors include norharman, rosmarinic acid, and alpha-methyl-tryptophan.

The term "a 1 adenosine inhibitor" as used herein denotes a compound, such as an antibody, which is capable of inhibiting the activity of the protein encoded by the ADORA1 gene in humans. Known A1 adenosine inhibitors include 8-cyclopentyl-1, 3-dimethylxanthine, 8-cyclopentyl-1, 3-dipropylxanthine, 8-phenyl-1, 3-dipropylxanthine, crotiteine, BG-9719, tonapofylline, FK-453, FK-838, rolifylline and N-0861.

The term "A2A adenosine inhibitor" as used herein means a compound, such as an antibody, capable of inhibiting the activity of the protein encoded by the ADORA2A gene in humans. Known adenosine inhibitors of A2A include ATL-4444, istradefylline, MSX-3, Pributylene, SCH-58261, SCH-412,348, SCH-442,416, 2-butyl-9-methyl-8- (triazol-2-yl) purin-6-amine, VER-6623, VER-6947, VER-7835, viadenant, and ZM-241,385.

The term "A2B adenosine inhibitor" as used herein denotes a compound, such as an antibody, which is capable of inhibiting the activity of the protein encoded by the ADORA2B gene in humans. Known adenosine inhibitors of A2B includeN- [5- (1-cyclopropyl-2, 6-dioxo-3-propyl-7)H-purin-8-yl) pyridin-2-yl]-N-ethylpyridine-3-carboxamide, 3-ethyl-1-propyl-8- [1- [ [3- (trifluoromethyl) phenyl ] methyl ester]Methyl radical]Pyrazol-4-yl]-7H-purine-2, 6-dione, MRS-1706, MRS-1754,N- [2- [ [ 2-phenyl-6- [4- (3-phenylpropyl) piperazine-1-carbonyl]-7H-pyrrolo [2,3-d]Pyrimidin-4-yl]Amino group]Ethyl radical]Acetamide, PSB-603, PSB-0788 and PSB-1115.

The term "A3A adenosine inhibitor" as used herein denotes a compound, such as an antibody, which is capable of inhibiting the activity of the protein encoded by the ADORA3 gene in humans. Known A3A adenosine inhibitors include KF-26777, MRS-545, MRS-1191, MRS-1220, MRS-1334, MRS-1523, MRS-3777, MRE-3005-F20, MRE-3008-F20, PSB-11, OT-7999, VUF-5574, and SSR 161421.

The term "arginase inhibitor" as used herein denotes compounds such as antibodies capable of inhibiting the activity of the protein encoded by the ARG1 or ARG2 gene in humans. Known arginase inhibitors include (2S) - (+) -amino-5-iodoacetamidopentanoic acid, NG-hydroxy-L-arginine, (2S) - (+) -amino-6-iodoacetamidohexanoic acid and (R) -2-amino-6-borono-2- (2- (piperidin-1-yl) ethyl) hexanoic acid.

The term "HDAC inhibitor" as used herein means a compound, such as an antibody, capable of inhibiting the activity of a protein that is a member of the histone deacetylase class, e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT 7. Known HDAC inhibitors include valproic acid, SAHA and romidepsin.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Brief Description of Drawings

FIG. 1 is a 2- [3- [ (3) prepared using zinc chloride as the source of counter ionsR) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]XRPD diffractogram of amorphous zinc salt of acetic acid (compound 1).

Fig. 2 shows PLM images of amorphous zinc salts of compound 1 prepared using zinc chloride as the source of counter ions.

Fig. 3 is a TG/DTA thermogram of the amorphous zinc salt of compound 1 prepared using zinc chloride as the source of counter ions.

Figure 4 shows the DSC thermogram for the amorphous zinc salt of compound 1 prepared using zinc chloride as the source of counter ions (top: first thermal cycle; bottom: second thermal cycle).

FIG. 5 is an amorphous zinc salt of Compound 1 prepared using zinc chloride as the source of the counterion¹H-NMR Spectroscopy (solvent: CDCl)₃)。

FIG. 6 is an amorphous zinc salt of Compound 1 as a source of counterion¹⁹F-NMR (solvent: CDCl)₃) Spectra.

Figure 7 is an FTIR spectrum of compound 1 in free form.

Fig. 8 is an FTIR spectrum of a 1:1 (w/w) mixture of the free form of compound 1 and the amorphous zinc salt of compound 1.

Fig. 9 is an FTIR spectrum of the amorphous zinc salt of compound 1.

FIG. 10 is an FTIR spectrum of ZnO.

FIG. 11 is ZnCl₂FTIR spectra of (1).

Detailed Description

To identify 2- [3- [ (3) having improved processability and stabilityR) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]Salt of acetic acid (compound 1), an LXR agonist, the inventors of the present invention conducted extensive salt screening experiments using over 65 different sources of counter ions and solvent systems prepared from over 15 solvents or solvent mixtures.

In the initial screening of common sources of counterions, the only salts prepared in the apparent crystal form were those prepared using hydrochloric acid, DL-mandelic acid and naphthalenesulfonic acid, the hydrochloride salt being the only salt that can be reproduced and scaled up. However, the hydrochloride salt was found to be associated with several strong acid salts (i.e., H)₂SO₄HBr, p-toluene sulfonic acid and methane sulfonic acid salts) tend to be unstable, e.g., the strong acid salts are found to readily undergo significant esterification with alcohol solvents. Subsequent studies of salts prepared with weak acids (e.g., oleic acid, caprylic acid, and acetic acid) have resulted in the isolation of only the free compounds.

Further, the inventors have found that the hydrochloride salt is unstable under vacuum and at moderate to high temperatures (e.g., above 50 ℃). Under these conditions, the hydrochloride salt tends to discolor and/or lose hydrochloric acid. The inventors have also found that extraction of the hydrochloride salt with water results in loss of hydrochloric acid, making further processing difficult. It has further been found that the hydrochloride salt undergoes significant esterification in the presence of a free alcohol group (e.g., when in an alcoholic solvent or in a formulation of an excipient having a free alcohol group such as sorbitol), or esterification in the presence of a lipophilic ester such as linoleate. These instability problems of the hydrochloride salt make it less suitable for processing into pharmaceutical products, and therefore salt screening studies were conducted to find a more suitable salt form.

Subsequent salt screening studies were conducted to identify counterion and solvent systems that favor the formation of salts with improved physical properties and stability. From these salt screening studies, including experiments with over 65 different sources of counter ion, it was found that the 2:1 (compound 1: zinc) salt was surprisingly one of the few salts formed as stable solids, unlike gels, gums, oils and semisolids formed with most sources of counter ion. As reproducible flowable solids, the 2:1 (compound 1: zinc) salts are more suitable for scale-up and development of pharmaceutical products.

2- [3- [ (3R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl ] methyl ester]Methyl- (2, 2-diphenylethyl) amino]Butoxy oxygen Base of]Phenyl radical]Acetic acid

2-[3-[(3R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]Acetic acid (compound 1) is an LXR β agonist with the following structure:

compound 1

Compound 1-mediated activation of the LXR signaling pathway has been shown to induce expression of ApoE, which acts as a tumor suppressor by virtue of its ability to modulate key features of tumorigenesis. Compound 1 has higher specificity for LXR β isoforms. These features include inhibition of cancer cell invasion (about 45% in vitro), inhibition of endothelial recruitment (about 50% in vitro), and reduction of circulation (about 40%) and tumors: (b)>60%) MDSC in the cells. In vitro studies, compound 1 induced ApoE gene expression 3-fold in cancer cells and up to 40-fold in human peripheral blood mononuclear cells (hpbmcs) compared to control cells. EC for ApoE-induced Compound 1₅₀385 nM in cancer cells and 271 nM in hBMC.

Compound 1 inhibited primary tumor growth by 48-95% in mouse tumor models of melanoma (carrying different genetic backgrounds), glioblastoma, TNBC, ovarian and lung cancers isogenic and human xenografts. The extent of tumor growth inhibition varies with the model. Compound 1 inhibited metastatic spread of cancer cells by a factor of 9 in a mouse model of TNBC metastasis. Furthermore, the anti-tumor activity of compound 1 in combination with the anti-PD-1 antibody inhibited tumor growth by >80% in an isogenic mouse melanoma model that was not otherwise responsive to the anti-PD-1 antibody. Furthermore, when mice received a combination therapy of compound 1 and anti-CTLA-4 antibody, inhibition of tumor growth in the same syngeneic mouse melanoma model was superior compared to either therapy alone. Similarly, compound 1 showed superior antitumor efficacy (>80%) in combination with dacarbazine in an isogenic mouse melanoma model compared to either treatment alone. In tumor growth studies, the lowest effective dose in the range of 25-40 mg/kg/day administered orally (PO) resulted in exposures in the range of 10,000 and 50,000 ng-h/mL.

In the safety pharmacology evaluation, compound 1 produced a significant increase in human ether-a-go-go related gene (hERG) channel conductance in the in vitro hERG assay, but no inhibition. There was no compound 1-related effect on qualitative Electrocardiogram (ECG) parameters (PR or QTc interval or QRS duration) in dogs, but there was a dose-related reduction in mean heart rate on day 1 of the post-dose interval, which differed significantly in females after a 150 (step down to 100) mg/kg/day dose. This change was not observed during the recovery period and was not considered detrimental. In addition, adverse effects were not noted during neurobehavioral function observation pools (FOB) or respiratory evaluation in rats. The potential for cardiovascular, respiratory or Central Nervous System (CNS) system effects is considered low in view of the favorable safety profile of compound 1 at the highest dose tested in repeated dose toxicity studies.

In the oral PK study, compound 1 was well absorbed in CD-1 mice, where the calculated absolute oral bioavailability (% F) was often>100%, indicating possible enterohepatic recirculation of the parent compound. Time to maximum plasma concentration (T)_max) Similar for males and females and in the 2 to 8 hour range. The mean apparent oral half-life (t provides) in mice is in the range of 6.5 to 8 hours. There was a significant food effect indicating that compound 1 plasma concentrations were high when administered to mice in the fed state>2 times. Average% F combined in Sprague-Dawley rats (in 2- [3- [ (3))R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl ] carbonyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy group]Phenyl radical]After 30 mg/kg oral dose of acetic acid) was moderate (about 31%), and T_maxThis is similar for males and females ranging from 4 to 8 hours. Compound 1 is cleared at a lower rate in female rats than in males, which results in higher systemic exposure of compound 1 in females at all dose levels tested. The mean apparent oral t, in female rats was 6.5 hours (not calculable in males). In male beagle dogs providing oral doses of Compound 1, T_maxRanging from 4 to 8 hours. Mean% F is moderate (18-30%, depending on dose and formulation) and mean apparent oral t North in the 5 to 6.7 hour range. In cynomolgus monkeys, the average% F is low to moderate (6-19%, depending on dose and formulation). After oral dose, monkeys had a mean T of 4 hours_max. The mean oral t, has a range of 5.5-8 hours.

Compound 1 undergoes phase I and phase II metabolism, which includes oxidation, dealkylation, glucuronidation, and combinations thereof. In vitro, compound 1 is metabolized primarily by the cytochrome P450 (CYP) isoform, CYP3a4, but it is also a substrate for CYP2E1, CYP2C9, CYP2C19, and possibly CYP2J 2. Although compound 1 is not a strong inhibitor of any human CYP450 in vitro, it is a moderate inhibitor of CYP2C8 (7.5 μ M50% inhibitory concentration [ IC50]) and a weak inhibitor of 2B6 (15 μ M IC 50). Compound 1 inhibited 1a1, 2a6, 2C9, 2C19, 2D6, 2E1, and 3a4 very weakly in vitro, but CYP3A time-dependent inhibition (TDI) was demonstrated in vitro using testosterone as a substrate. CYP3A induction by compound 1 and the potential of CYP2B6 induction by compound 1 was demonstrated in cryopreserved primary cultures of hepatocytes (2 donors) (1 concentration in 1/3 donors was induced > 2-fold). Compound 1 did not induce CYP1a 2. In the efflux transporter, compound 1 did not inhibit P-glycoprotein (P-gp) in vitro, but inhibited Breast Cancer Resistance Protein (BCRP) trafficking (55% at 5 μ M). Compound 1 is a potent inhibitor of uptake of transporter Organic Anion Transport Polypeptide (OATP) 1B1 in vitro (0.099 μ M IC 50). Compound 1 also appears to be a moderate inhibitor of OATP1B3 (3.7 μ M IC 50). Compound 1 only weakly inhibited OAT1, OAT3 and OCT2 in vitro with less than 50% inhibition at 50 μ M.

Based on animal toxicology studies, potential risks of using compound 1 in clinical settings include elevated serum cholesterol and TG, neutropenia/leukopenia, nausea and/or vomiting, elevated liver enzymes, development or worsening of cataracts, cardiac rhythm disorders and/or reduced cardiac function, and the like,

Paralacrimal adenocarcinoma and/or generalized edema.

Nivolumab

Nivolumab is a fully human immunoglobulin (Ig) G4 monoclonal antibody directed against negative immunomodulatory human cell surface receptor programmed death-1 (PD-1) with immune checkpoint inhibitory and anti-tumor activity. Nivolumab binds and blocks activation of PD-1, an Ig superfamily transmembrane protein, by its ligand programmed cell death ligand 1 (PD-L1) overexpressed on certain cancer cells and programmed cell death ligand 2 (PD-L2) expressed predominantly on antigen presenting cells. This results in the activation of T cells and a cell-mediated immune response against tumor cells or pathogens. Activated PD-1 down-regulates T-cell activation and plays a key role in tumor escape from host immunity. The nivolumab dose will be 240 mg administered as an intravenous infusion over 60 minutes on

days

1 and 15 of each 28-day cycle.

There is the potential for overlapping toxicity between compound 1 and nivolumab. In particular, DLT for grade 4 neutropenia has been seen on compound 1 single agent therapy, and myelosuppression can be observed with nivolumab treatment. The pharmacological effects of compound 1 include modulation of sterol biosynthesis. Thus, hyperlipidemia has been observed in subjects treated with compound 1, which is also reported in subjects treated with nivolumab. Liver function abnormalities have been observed in preclinical toxicity studies using compound 1, and immune-mediated hepatitis has been observed with nivolumab treatment.

Immunobio monocistron

Ipilimumab is a recombinant human IgG1 kappa monoclonal antibody that binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). CTLA-4 is a negative regulator of T-cell activity. By binding to CTLA-4, the IL-4 block the interaction of CTLA-4 with its ligand CD80/CD 86. Blockade of CTLA-4 has been shown to enhance T-cell activation and proliferation, including activation and proliferation of tumor infiltrating T-effector cells. Inhibition of CTLA-4 signaling can also reduce T-regulatory cell function, which can contribute to an overall increase in T-cell responsiveness (including anti-tumor immune responses).

In certain embodiments, the ipilimumab dose is 3 mg/kg, up to 4 doses, administered as an intravenous infusion on day 1 of each 28 day cycle.

There is the potential for overlapping toxicity between compound 1 and the ipilimumab. Liver function abnormalities have been observed in preclinical toxicity studies using compound 1; immune-mediated hepatitis has been observed with the use of ipilimumab therapy.

Docetaxel

Docetaxel is an antitumor agent belonging to the taxane family. It is prepared by semi-synthesis starting from precursors extracted from renewable coniferous biomass of the taxus species. Docetaxel has the chemical name of (2R,3S) -N-carboxy-3-phenylisoserine, N-tert-butyl ester, and 13-ester, trihydrate of 5 b-20-epoxy-1, 2a,4,7b, 10b, 13 a-hexahydroxy-taxus-11-en-9-one 4-acetate 2-benzoate.

In certain embodiments, docetaxel is administered as an intravenous infusion on

days

1, 8, and 15 of each 28-day cycle. In certain embodiments, the docetaxel dose is 35 mg/m². In certain embodiments, 28 mg/m²The dosage of docetaxel.

There is a potential for overlapping toxicity between compound 1 and docetaxel. In particular, DLT for grade 4 neutropenia has been seen on compound 1 single agent therapy, and myelosuppression can be observed with docetaxel treatment. Liver function abnormalities have been observed in preclinical toxicity studies using compound 1; hepatotoxicity has been observed with docetaxel treatment.

Pembrolizumab

Pembrolizumab is an antibody that blocks programmed death receptor-1 (PD 1). Pembrolizumab is a humanized monoclonal IgG4 kappa antibody with an approximate molecular weight of 149 kDa. Pembrolizumab was produced in recombinant Chinese Hamster Ovary (CHO) cells.

In certain embodiments, pembrolizumab is administered at a dose of 200 mg using a 30 minute intravenous infusion on day 1 of each 21-day cycle after all procedures and evaluations have been completed and prior to the administration of other drugs, with a 30 minute interval between the administration of the next drug.

Carboplatin

The chemical name of carboplatin (USP) is platinum, diamine [1, 1-cyclobutane dicarboxylate (2-) -O,O′]-，(SP-4-2). Carboplatin (USP) is a crystalline powder. It is soluble in water at a concentration of about 14 mg/mL and the pH of a 1% solution is from 5 to 7. It is substantially insoluble in ethanol, acetone and dimethylacetamide. Carboplatin predominantly produces interchain DNA cross-links rather than DNA-protein cross-links. This effect is clearly cell cycle non-specific. Carboplatin induces an equal number of drug-DNA cross-links, resulting in equivalent lesions and biological effects.

In certain embodiments, the initial dose of carboplatin injection is determined by using a mathematical formula that is based on the subject's pre-existing renal function or renal function and the desired platelet nadir (since renal excretion is the primary pathway for elimination of carboplatin). In contrast to empirical dose calculations based on body surface area, application of the quantitative administration formula allows compensation for subject variations in pre-treatment renal function that might otherwise result in under-dosing (in subjects with over-average renal function) or over-dosing (in subjects with an injury to renal function).

A simple formula for calculating the dose based on the subject's glomerular filtration rate (GFR, mL/min) and the carboplatin injection target area under the concentration versus time curve (AUC, mg/ml.min) has been proposed by Calvert. In these studies, GFR was measured by Cr-EDTA clearance. The Calvert formula for carboplatin dosing is as follows:

total dose (mg) = (target AUC) × (GFR +25)

It should be noted that with this formula, in mg, not mg/m²Calculating the total of carboplatin for the unitAnd (4) dosage. The target AUC of 4 mg/ml.min to 6 mg/ml.min using the single agent carboplatin appears to provide the most appropriate dose range in previously treated subjects. This study also demonstrated a trend between AUC for the single agent carboplatin administered to previously treated subjects and the likelihood of toxicity.

Pemetrexed

Pemetrexed (for injection) is a folate analogue metabolic inhibitor. The pharmaceutical substance pemetrexed disodium heptahydrate has the chemical name L-glutamic acid,N- [4- [2- (2-amino-4, 7-dihydro-4-oxo-1)H-pyrrolo [2,3-d]Pyrimidin-5-yl) ethyl]Benzoyl radical]-, disodium salt, heptahydrate, having C₂₀H₁₉N₅Na₂O₆•7H₂The molecular formula of O and a molecular weight of 597.49 g/mol.

Pemetrexed exerts its anti-tumor activity by disrupting folate-dependent metabolic processes essential for cell replication. In vitro studies have demonstrated that pemetrexed behaves as a multi-target antifolate by inhibiting Thymidylate Synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT), which are critical for de novo biosynthesis of thymidine and purine nucleotides. Polyglutamate metabolites of pemetrexed with extended intracellular half-life lead to prolonged pemetrexed drug effects in malignant cells.

In certain embodiments, the pemetrexed dose is 500 mg/m on day 1 of each 21-day cycle²And a maximum of 4 cycles. In certain embodiments, subjects treated with pemetrexed must be instructed to take folic acid and vitamin B12 as a preventative measure to reduce treatment-related hematologic and GI toxicity. In certain embodiments, the subject may also be prescribed corticosteroids for 2 administrations per day for 3 days, beginning the day prior to each treatment with pemetrexed.

There is the potential for overlapping toxicity between compound 1, pemetrexed and carboplatin. In particular, DLT for grade 4 neutropenia has been seen on compound 1 single agent therapy and myelosuppression is observed with pemetrexed in combination with compound 1 and carboplatin.

Adverse events

An Adverse Event (AE) is any adverse medical event in the subject to whom the drug product is administered or in a subject under clinical study, and it does not necessarily have to have a causal relationship to this treatment. Thus, an AE may be any adverse and unexpected sign (including abnormal laboratory findings), symptom, or disease temporally associated with the use of a study product, whether or not associated with the study product.

Death and Progressive Disease (PD) were not considered AEs. Death is considered to be the result of one or more major AEs, and PD is considered to be worsening of the underlying disease. Pre-existing disorders (present prior to the start of the AE collection period) are considered to be concurrent medical disorders and not AEs. However, the exacerbation or complication of such a complication is AE.

An AE or suspected adverse reaction is considered severe in the following cases: it leads to death; is life-threatening, i.e., the subject is at immediate risk of dying from the reaction as it occurs, but does not include the assumption that reactions that may cause death have occurred in a more severe form; requiring hospitalization of the subject or extending existing hospitalization; resulting in persistent or significant disability/disability; is a congenital abnormality/birth defect; or as an important medical event.

Method of treatment

The methods described herein may be used to treat cancer.

Treating cancer can result in a reduction in the size or volume of the tumor. For example, after treatment, the tumor size is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) relative to its size prior to treatment. The size of the tumor can be measured by any reproducible measurement method. The size of the tumor can be measured as the diameter of the tumor or by any reproducible measurement method.

Treatment of cancer can further lead to a reduction in the number of tumors. For example, after treatment, the number of tumors is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to the number prior to treatment. Tumor numbers can be measured by any reproducible measurement method. Tumor number can be measured by counting tumors that are visible to the naked eye or at a specified magnification (e.g., 2x, 3x, 4x, 5x, 10x, or 50 x).

Treatment of cancer can result in a reduction in the number of metastatic nodules in other tissues or organs distant from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to the number prior to treatment. The number of metastatic nodules can be measured by any reproducible measurement method. The number of metastatic nodules can be measured by counting metastatic nodules visible to the naked eye or at a specified magnification (e.g., 2x, 10x, or 50 x).

Treatment of cancer may result in an increase in the average survival time of a population of subjects treated according to the invention compared to an untreated population of subjects. For example, the average survival time increases by more than 30 days (more than 60 days, 90 days, or 120 days). The increase in the mean survival time of the population can be measured by any reproducible method. For example, by calculating the mean length of survival for a population after the start of treatment with a compound of the invention, the increase in mean survival time for the population can be measured. For example, by calculating the mean length of survival for a population after completion of a first round of treatment with a compound of the invention, the increase in mean survival time for a population can also be measured.

Treatment of cancer may also result in a decrease in mortality in the treated subject population compared to the untreated population. For example, mortality decreases by more than 2% (e.g., more than 5%, 10%, or 25%). The decline in mortality rate in the treated population of subjects can be measured by any reproducible method, for example, by calculating the average number of disease-related deaths per unit time for the population after starting treatment with a compound of the invention. For example, a decrease in mortality of a population can also be measured by calculating the average number of disease-related deaths per unit time after completion of the first round of treatment with a compound of the invention for the population.

Treatment of cancer may also result in an increased mean progression-free survival time for the treated population of subjects compared to the untreated population. For example, the mean progression-free survival time is increased by more than 30 days (more than 60, 90 or 120 days). The increase in mean progression free survival time of the population can be measured by any reproducible method. For example, by calculating the mean progression free survival length after starting treatment with a compound of the invention for a population, the increase in the mean progression free survival time for the population can be measured. For example, by calculating the mean length of progression free survival for a population after completion of a first round of treatment with a compound of the invention, the increase in mean progression free survival time for a population can also be measured.

Composition comprising a metal oxide and a metal oxide

Within the scope of the present invention are compositions comprising a suitable carrier and one or more of the above-described therapeutic agents.

As noted above, the pharmaceutical compositions of the present invention additionally include pharmaceutically acceptable excipients, as used herein, including any or all of solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants suitable for the particular intended dosage form. Remington's Pharmaceutical Sciences, sixteenth edition, e.w. Martin (Mack Publishing co., Easton, Pa., 1980) discloses various excipients used in formulating Pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional excipient is incompatible with the compounds of the present invention (such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition), its use is contemplated within the scope of the present invention.

The above compositions in any of the above forms may be used to treat cancer or any other disease or disorder described herein. An effective amount means the amount of active compound/agent required to provide a therapeutic effect to the subject being treated. As will be appreciated by those skilled in the art, effective dosages will vary with the type of condition being treated, the route of administration, excipient usage, and the possibility of co-use with other therapeutic treatments.

The pharmaceutical compositions of the present invention may be administered parenterally, orally, nasally, rectally, topically or buccally. The term "parenteral" as used herein means subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection, and any suitable infusion technique.

Sterile injectable compositions can be solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent.

Compositions for oral administration may be in any orally acceptable dosage form, including capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring or coloring agents may be added.

Pharmaceutical compositions for topical administration according to the described invention may be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols or oils. Alternatively, the topical formulation may be in the form of a patch or dressing impregnated with the active ingredient, which may optionally contain one or more excipients or diluents. In certain preferred embodiments, the topical formulations comprise materials that will enhance the absorption or penetration of the active agent through the skin or other affected area.

Combination therapy

In certain embodiments of the methods described herein, the pharmaceutical composition may further comprise an additional compound having antiproliferative activity.

It is also understood that the compounds and pharmaceutical compositions of the present invention can be formulated and used in combination therapy, that is, the compounds and pharmaceutical compositions can be formulated together or administered concurrently before or after, or in parallel with, one or more other desired therapeutic agents or medical procedures (e.g., surgery and/or radiation therapy). The particular combination of treatments (therapies or procedures) employed in a combination regimen will take into account the compatibility of the desired therapy and/or procedure with the desired therapeutic effect to be achieved. It is also understood that the therapies employed may achieve the desired effect on the same disorder, or they may achieve different effects (e.g., control of any adverse effects).

Examples

Example 1. method

X-ray powder diffraction (XRPD)

Method 1

XRPD analysis was performed on PANalytical X' pert pro, scanning the sample between 3 and 35 ° 2 θ. The material was gently ground to release any agglomerates and loaded onto a multi-well plate with a Mylar polymer film to hold the sample. The multi-well plate was then placed in a diffractometer and analyzed using Cu K radiation (α 1 λ = 1.54060A; α 2 = 1.54443 a; β = 1.39225 a; α 1: α 2 ratio = 0.5) (run in penetration mode (step size 0.0130 ° 2 θ, using a 40 kV/40 mA generator setting).

Method 2

X-ray powder diffraction patterns were obtained using Bruker D8 Advance equipped with a Cu ka radiation source (λ =1.54 ° a), a 9-position sample holder, and a LYNXEYE overspeed detector. The sample was placed on a 0 background silicon plate rack.

Polarized Light Microscopy (PLM)

Method 1

The presence of birefringence was determined using an Olympus BX53 polarization microscope equipped with a Motic camera and image capture software (Motic Images Plus 3.0). All images were recorded using either a 10x or 20 x objective lens.

Method 2

The samples were analyzed using an Olympus BX53 polarizing light microscope equipped with a PAXcam 3 digital microscope camera.

Thermogravimetric/differential thermal analysis (TG/DTA)

Method 1

Approximately 5 mg of material was weighed into an open aluminum pan and loaded into a thermogravimetric/differential thermal analyzer (TG/DTA) and maintained at room temperature. The sample was then heated from 20 ℃ to 300 ℃ at a rate of 10 ℃/min under a nitrogen purge, and the change in sample weight and any differential thermal events (DTA) were recorded during this time.

Method 2

TGA data was collected using TA Instruments TGA Q500. Typically, the sample (about 10 mg) is placed in an open pre-tared aluminum sample pan and scanned at 25-300 ℃ using a 60 mL/min nitrogen purge at a rate of 10 ℃/min.

Differential Scanning Calorimetry (DSC)

Method 1

Approximately 5 mg of material was weighed into an aluminum DSC pan and sealed with an aluminum lid. The sample pans were then loaded into a TA Instruments Discovery DSC 2500 differential scanning calorimeter equipped with an RC90 cooler. Once a stable heat flow response was obtained, the sample and reference were heated/cooled under a nitrogen purge using the following temperature program and the resulting heat flow response monitored.

Procedure 1

Heating at 10 ℃/min to 20 ℃ to 260 ℃ and holding at 260 ℃ for 3 minutes;

cooling at 10 ℃/min between 260 ℃ and 20 ℃, holding at 20 ℃ for 3 minutes;

cooling at 10 ℃/min between 260 ℃ and 20 ℃, holding at 20 ℃ for 3 minutes.

Procedure 2

Heating at 10 ℃/min to 20 ℃ to 270 ℃ and holding at 270 ℃ for 3 minutes;

cooling at 10 ℃/min from 270 ℃ to 20 ℃, holding at 20 ℃ for 3 minutes;

cool 270 ℃ to 20 ℃ at 10 ℃/min, hold at 20 ℃ for 3 min.

Method 2

DSC data were collected using a TA Instruments Q10 DSC. Typically, samples (2 to 8 mg) were placed in unsealed but hermetically covered albutanized aluminum sample pans and scanned from 30 to 300 ℃ at a rate of 10 ℃/min under a nitrogen purge of 50 mL/min.

¹ H nuclear magnetic resonance spectroscopy ( ¹ H-NMR)

Method 1

The procedure was carried out on a Bruker AV500 (frequency: 500 MHz)¹H-NMR spectroscopy experiment. In CDCl₃The experiment was performed and each sample was prepared at a concentration of about 10 mM.

Method 2

Samples were prepared by dissolving the compound in deuterated dimethylsulfoxide containing 0.05% (v/v) Tetramethylsilane (TMS). Spectra were collected using TopSpin software on a Bruker Avance 300 MHz NMR at ambient temperature. For proton NMR, the number of scans was 16.

¹⁹ F nuclear magnetic resonance spectroscopy ( ¹⁹ F-NMR)

The procedure was carried out on a Bruker AV500 (frequency: 470 MHz)¹⁹F-NMR spectrum experiment.

In CDCl₃The experiment was performed and each sample was prepared at a concentration of about 10 mM.

High performance liquid chromatography-ultraviolet detection (HPLC-UV)

The HPLC method used a C18 column and an acetonitrile/water/trifluoroacetic acid gradient.

Dynamic Vapor Sorption (DVS)

The samples were analyzed using an Aquadyne DVS-2 gravimetric water uptake analyzer. The relative humidity was adjusted between 2-95% and the sample weight was continuously monitored and recorded.

Karl Fisher(KF)

The apparent water content in the samples was determined by Karl Fischer titration using a Mettler Toledo DL39 Coulometric KF titrimeter. HYDRANAL-Coulomat AD was used as titrant. About 20 mg of solid was used for titration. The analytical parameters are shown in table 1 below:

TABLE 1

KF parameter	Value of
		Rate [% ]]	40
Mixing time [ second]	10
		Automatic start	Whether or not
Blank [ mu g]	0
		Drift [ mu g/min]	5
Computing	Ug
		Standby	Is that
Initial drift [ mu g/min]	<10
		Initial potential [ mV]	100

Fourier transform Infrared Spectroscopy (FTIR)

FTIR analysis was performed on a Thermo Scientific, NICOLET IS 10 FTIR spectrometer with Attenuated Total Reflectance (ATR). Enough material was placed in the center of the plate of the spectrometer and the spectra were acquired.

Example 2 salt screening

Study 1

A first study was carried out to identify 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]-methyl- (2, 2-diphenylmethyl) amino]Butoxy radical]Phenyl radical]A salt of acetic acid. 26 sources of counterions were selected for 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]-methyl- (2, 2-diphenylmethyl) amino]Butoxy radical]Phenyl radical]And (4) screening salts of acetic acid. The source of counterions is listed in table 2 below:

TABLE 2 List of selected sources of counter ions for the salt screening experiments in study 1

Source of counterions	Numbering
		Hydrochloric acid (HCl)	1
Sulfuric acid (H)₂SO₄)	2
		Benzenesulfonic acid (C)₆H₅O₃SH)	3
Phosphoric acid (H)₃PO₄)	4
		Sodium hydroxide (NaOH)	5
Potassium hydroxide (KOH)	6
		Calcium hydroxide (Ca (OH)₂)	7
Maleic acid	8
		Fumaric acid	9
L-arginine	10
		L-histidine	11
L-malic acid	12
		Diethanolamine (DEA)	13
Salicylic acid	14
		Benzoic acid	15
DL-mandelic acid	16
		Ethylene diamine	17
Succinic acid	18
		L-tartaric acid	19
L-aspartic acid	20
		L-lysine	21
L-ascorbic acid	22
		Citric acid	23
Naphthalene-2-sulfonic acid	24
		1, 2-ethane-disulfonic acid	25
Hydriodic acid	26

Of the sources of counter ions studied, only three (hydrochloride, DL-mandelic acid and naphthalenesulfonic acid) provided crystalline salts, and their reproduction and scale-up was successful only for the hydrochloride salt.

Subsequent studies revealed that the hydrochloride salt was found and recovered from strong acids (e.g., H)₂SO₄HBr, p-toluenesulfonic acid and methanesulfonic acid) are unstable. In particular, these salts react with excipients and/or components of the formulation. This finding led to research into the use of weak acids (e.g., oleic acid, caprylic acid, and acetic acid) as sources of counterions, none of which is compatible with 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (tris)Fluoromethyl) phenyl]-methyl- (2, 2-diphenylmethyl) amino]Butoxy radical]Phenyl radical]Acetic acid forms a salt.

Study 2

A second study was conducted to identify salts that provide improved processability and stability. The 34 sources of counter ions and 12 solvent conditions studied in this study are summarized in tables 3 and 4 below:

TABLE 3 List of selected sources of counter ions for the salt screening experiments in study 2

TABLE 4 List of selected solvent conditions for the salt screening experiments in study 2

Solvents and solvent mixtures
	Isopropanol (IPA)
Tetrahydrofuran (THF)
	Dichloromethane (DCM)
Ethyl acetate (EtOAc)
	Heptane (Hep)
Tert-butyl methyl ether (TBME)
	Water (H)₂O)
1, 4-dioxane
	Acetone (II)
THF:H₂O (9:1)
	Acetone H₂O (9:1)
IPA:H₂O (95:5)

Initial solubility evaluations in 12 solvents/solvent mixtures were performed using amorphous compound 1. Dissolution was observed in most samples. Studies were performed by cooling, anti-solvent addition, maturation, liquid assisted milling, pure milling and evaporative salt screening. The isolated gums were subjected to a hot/cold maturation cycle (room temperature/50 ℃), sonication, long term storage under vacuum, long term storage at 40 ℃/75% RH, milling with anti-solvent, and liquid assisted milling (LAG) with water. From the extensive salt screening, 8 hits were obtained. Amorphous hits with high glass transition temperatures (Tg) were identified from zinc chloride, naphthalene-2-sulfonic acid, 1, 5-naphthalenedisulfonic acid and ethanesulfonic acid. One crystallization hit was identified from ethanesulfonic acid. The poorly crystalline material was separated from calcium hydroxide, magnesium hydroxide and aluminum hydroxide.

Because of the limited characterization of isolated hits, among the most promising counterions: focusing and screening are carried out on calcium hydroxide, aluminum hydroxide, ethane sulfonic acid and naphthalene-2-sulfonic acid. Calcium hydroxide and aluminum hydroxide were selected to evaluate whether the initial hits isolated via counterion exchange were single metal salts or mixed metal salts. The solids isolated from the additional screen were consistent with the initial hits. From the additional screening, these samples may be single metal salts. Because these hits were not mixed salts, limited mixed metal screens were performed using magnesium hydroxide and calcium hydroxide. These attempts were unsuccessful. Ethane sulfonic acid and naphthalene-2-sulfonic acid were selected for further screening. However, the initial hits were not reproduced in further screening attempts. The amorphous solid was isolated, however, with a Tg lower than that of the free compound (32 deg.C) and HCl salt (about 70 to 75 deg.C).

The zinc salt was isolated using the following procedure:

compound 1 (about 250 mg) was dissolved in THF (5 vol) at room temperature. The solution was dispensed into HPLC vials (75 μ l, about 15 mg in solution). The solution was held at 50 ℃ for 5 minutes and observations were recorded. The sample was treated with 1.0 equivalent of sodium and then with the selected equivalent of counterion (0.5 equivalents of zinc chloride). The sample was held at 50 ℃ for 10 minutes and then cooled to 5 ℃ at 0.1 ℃/min. The resulting solution was kept at 5 ℃ overnight and then stored at-20 ℃. After 5 days, the sample was still in solution and allowed to evaporate at ambient conditions. After evaporation the gum was obtained and treated with 10 volumes of heptane followed by sonication for 2 hours. The collected solid was analyzed by XRPD and found to be amorphous.

None of the salts isolated from this study was suggested for development due to the long complex isolation procedure and the tendency of compound 1 to form a gum. However, several salts (e.g., zinc and aluminum) produced encouraging results, i.e., suitable salts may be feasible and motivated for further research.

Study 3

A third salt screening study was performed. Using an approximation with more than 8 on a 100 mg scalepK _aOr having a base of less than 4pK _aThe acceptable strong acids of (a) attempt salt formation reactions.

In a first set of salt screening experiments, the source of counter ions shown in table 5 below was studied.

Table 5: list of selected sources of counter ions for the first set of salt screening experiments in study 3

Source of counterions	Molecular weight (g/mol)	pKa₁
			Ethane-1, 2-disulfonic acid	190.20	-2.1
Naphthalene-1, 5, disulfonic acid	332.26	-3.4
			Ethanesulfonic acid	110.13	-1.6
Nitric acid	63.02	-1.3
			Naphthalene-2-sulfonic acid	208.24	0.17
Zinc hydroxide	99.38	14
			Choline	121.18	>11.0
Benzathine	240.35	10.0
			Benzethylamine (Benthamine)	211.30	9.4
Ethanolamine	61.80	9.6
			Diethanolamine (DEA)	105.14	9.5
L-arginine	174.20	9.0

Briefly, 2- [3- [ (3)R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylmethyl) amino]-butoxy radical]Phenyl radical]Acetic acid was suspended/dissolved in six different solvent systems-isopropanol, acetone, cyclohexanone, methyl ethyl ketone, ethyl acetate and THF. The corresponding source of counter ions (1.0 eq.) is then added as a solution (where possible), or via neat addition. The solution is temperature cycled between two specific temperatures (e.g., 25 and 40 ℃). All experiments were monitored for precipitation/crystallization. In vials where crystallization/precipitation is not evident, cooling to sub-ambient temperature or iterative anti-solvent addition using a suitable anti-solvent is performed with or without cooling to sub-ambient temperature (e.g., 5 ℃). In a vial where crystallization/precipitation is insignificant upon cooling and/or addition of an anti-solvent, the solvent is removed by evaporation. Any solids were isolated by filtration and analyzed by XRPD. Mainly oils or gums are obtained.

A second salt screen was performed using the source of counter ions shown in table 6 below.

Table 6: list of selected sources of counter ions for the second set of salt screening experiments in study 3

Source of counterions	Molecular weight (g/mol)	pKa₁
			Ethanedisulfonic acid sodium salt	234.16	-2.1
Thiocyanic acid	59.09	-1.3
			Dioctyl sulfosuccinic acid	422.16	-0.8
Naphthalenesulfonic acid sodium salt	230.22	0.2
			Dichloroacetic acid	128.94	1.3
Hydroxyethyl sodium sulfonate	148.11	1.7
			Pamoic acid	388.38	2.5
Hippuric acid	179.18	3.6
			L-lysine	146.19	9.2
Histidine	155.15	9.2
			1- (2-hydroxyethyl) -pyrrolidine	115.18	9.4

The procedure for preparing the salts using the source of counter ions of table 5 is as follows. 2- [3- [(3R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylmethyl) amino]Butoxy radical]-phenyl radical]Acetic acid is produced from its hydrochloride salt according to known procedures (Collins et al J. Med. chem., 2002, 45: 1963-. 100 mg of free material was transferred into a 20 mL vial. Ethyl acetate (1 mL) was added to each vial followed by 1.0 equivalents of each source of counter ions. The samples were temperature cycled between ambient and 40 ℃ for a4 hour period and stirred for 24 hours. Heptane was added as an anti-solvent to any clear solution and the sample was placed in a refrigerator for 24 hours. All the solids so obtained were analyzed by XRPD and all samples still as clear solutions were uncapped and allowed to evaporate. These sources of counterions produce solids or viscous gel-like solids that are identified as sources of counterions.

Another salt screen was performed using an aqueous precipitation protocol. The salt screen uses a metathesis procedure in which a 1:1 sodium salt of compound 1 is prepared and 1.0, 0.5, or 0.3 equivalent of the selected metal counterion is added as its chloride or sulfate. Alternatively, free compound 1 is reacted with the selected metal counter ion as its acetate or sulfate. These aqueous precipitations produced free-flowing white solids from zinc, aluminum and bismuth.

The counterions shown in table 7 below were investigated.

Table 7: list of selected sources of counter ions for the third set of salt screening experiments in study 3

Source of counterions
	Zinc chloride
Bismuth chloride
	Zinc acetate
Aluminium sulphate

The zinc salt was prepared via metathesis using the following procedure.

Compound 1 (200 mg) was dissolved in methanol (2 mL) to form a mixture. A 1M NaOH solution (336 µ L, 1.0 equivalents) was then added to the mixture to form the 1:1 sodium salt of compound 1. Zinc chloride (22.5 mg, 0.5 eq) was dissolved in water (1 mL) and the resulting solution was added to the mixture, after which a white solid precipitated out of solution. Additional water (1 mL) was added to the mixture and the reaction vessel was sealed with parafilm, after which the mixture was temperature cycled between ambient and 40 ℃ for 24 hours with stirring in 4 hour periods. Methanol was removed from the mixture via rotary evaporation and water (10 mL) was added to reduce solubility. The resulting precipitate was separated via Buchner filtration (Whatman grade 1 filter paper, full leaf = 55 mm) and washed with water (100 mL) to remove any remaining sodium chloride. The solid was dried on filter paper to yield a white powder (152.9 mg, 72%).

The material obtained via the above salt metathesis procedure was subjected to the method described in example 1By ICP, XRPD (FIG. 1), PLM (FIG. 2), TG/DTA (FIG. 3), DSC (FIG. 4),¹H-NMR (FIG. 5) and¹⁹F-NMR (FIG. 6). Characterization of the substance yields the following information:

the material contained 5.27 wt% zinc by ICP analysis. The stoichiometric salt is 9.9 wt%, so about 0.51 equivalents of zinc (expected 0.5 equivalents) are present.

The material contained 0.19 wt% sodium by ICP analysis. The stoichiometric salt is 3.7 wt%, so about 5% of the material is the 1:1 sodium salt and about 95% of the material is the 2:1 zinc salt.

The material was amorphous by XRPD analysis.

The material showed some birefringence with irregular morphology by PLM analysis.

There is minimal mass loss in the TG trace due to decomposition.

By DSC, there is an endothermic event, with a starting temperature of 51 ℃ in the first thermal cycle and a steep event corresponding to a glass transition, with a midpoint temperature of 52 ℃ in the second thermal cycle.

•¹The H-NMR spectrum was consistent with that of Compound 1.

•¹⁹F-NMR showed one main peak.

The purity of the amorphous as determined by HPLC analysis is shown in table 8 below.

Table 8: purity and stability of zinc salt of compound 1 prepared from zinc chloride

Time (sky)	Purity of
		0	99.3%
7 (storage at 40 ℃/75% RH)	99.5%
		7 (stored at 80 ℃ C.)	96.2%

As shown in table 8, the zinc salt prepared by the above method is stable after storage at 40 ℃/75% RH and at 80 ℃ for 1 week.

Zinc acetate was also used as the source of the counterion to make the zinc salt. Briefly, Compound 1 (78 mg) was dissolved in methanol (1.5 mL) and zinc acetate was dissolved in methanol/water (9:1; 1.5 mL), after which the two solutions so obtained were combined to form a mixture. A white solid formed immediately and precipitated out of solution. The sample was then cycled between ambient temperature and 40 ℃ for about 5 hours, after which a small amount of white solid remained undissolved. The solvent was then removed via rotary evaporation, and methanol (5 mL) was added to the solid. The cycle and solvent evaporation steps were repeated, then methanol/water (9:1; 5 mL) was added to the sample, and the cycle and solvent evaporation steps were repeated once more. The samples were then freeze-dried overnight to remove water, after which a white solid was obtained.

The following procedure was used to prepare the aluminum salt via metathesis. Compound 1 (200 mg) was dissolved in methanol (2 mL). The sodium salt was prepared by adding 1M NaOH stock solution (336 µ L, 1.0 eq). Methanol was removed via rotary evaporation. Aluminum sulfate and water (2 mL) were added and the samples were temperature cycled between ambient and 40 ℃ for 24 hours in 4 hour cycles. The solids were separated via centrifugation. Water (3 mL) was added and the slurry was stirred at ambient temperature for 18 hours. The solid was filtered using a buchner funnel (Whatman grade 1 filter paper, ca = 42.5 mm) and washed with water (100 mL) and then dried on the filter paper for 10 minutes. The solid was dried under vacuum at ambient temperature for about 2 hours to yield a white powder (128.7 mg, 61%).

Example 3 FTIR analysis

Study of

Preparation of free Compound 1 and Zinc salt in CHCl₃In (1)Stock solutions. In a volumetric flask, the free form of Compound 1 (50 mg) was taken up in CHCl₃Dilute to 10 mL. The zinc salt of Compound 1 (50 mg) was also taken up in a separate volumetric flask with CHCl₃Dilute to 10 mL. FTIR spectra were collected according to the FTIR method described in example 1, for the free form of compound 1 (see fig. 7) and the zinc salt form of compound 1 (see fig. 9). FTIR spectra were then taken of samples with increasing concentrations of the zinc salt of compound 1, such as:

● 1 free form of Compound 1A 1:1 (w/w) mixture of zinc salt (mixture of free form stock solution (500 μ L) and zinc salt stock solution (500 μ L)) (see FIG. 8);

● 1 free form of Compound 1A 1:4 (w/w) mixture of zinc salt (mixture of free form stock solution (200 μ L) and zinc salt stock solution (800 μ L));

● 1 free form of Compound 1A 1:9 (w/w) mixture of zinc salt (mixture of free form stock solution (100 μ L) and zinc salt stock solution (900 μ L));

● and Compound 1 in free form A5: 95 (w/w) mixture of zinc salts (mixture of free form stock solution (50 μ L) and zinc salt stock solution (950 μ L)).

The spectra were compared to each other and to ZnO (FIG. 10) and ZnCl₂Comparison of FTIR spectra (FIG. 11).

The free form of Compound 1 appears at about 1710 cm^-1A unique characteristic peak at (see fig. 7), which is absent in the zinc salt spectrum (see fig. 9). Observed at 1710 cm^-1The peak at (a) shrinks as the percentage of free form decreases. Furthermore, a low intensity of about 1590 cm was observed in the free compound^-1Broad peak at (a), but as the zinc salt concentration increases, its intensity increases.

Passing through a tube at 1710 cm^-1The disappearance of the IR peak at (a) confirms the zinc salt, presumably due to the coordination of the carboxylate carbonyl group with the zinc. Furthermore, the zinc salt is not contained in ZnO (characterized by being at 605 cm)^-1Is started to< 500 cm^-1Unique large broad band) or ZnCl₂(characterized in that the length of the groove is 3386 cm^-1Unique broad peak at (b) to further confirm that the zinc salt is not presentSimply a mixture of the free compound and inorganic zinc.

Other embodiments

All documents and similar materials cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such documents and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated documents and similar materials differ or contradict this application (including but not limited to defined terms, usage of terms, described techniques, etc.), the present application controls.

While the methods have been described in connection with various embodiments and examples, there is no intent to limit the methods to such embodiments or examples. On the contrary, the present disclosure encompasses various alternatives, modifications, and equivalents, as will be apparent to those skilled in the art.

While the method has been particularly shown and described with reference to specific exemplary embodiments, it will be understood that various changes in form and detail may be made therein without departing from the spirit and scope of the disclosure. Therefore, it is intended to claim all embodiments and equivalents thereof within the scope and spirit of the present disclosure. The claims, descriptions, and diagrams of the methods, systems, and assays of the present disclosure should not be construed as limited to the order of the elements described unless stated to that effect. This application claims the benefit of U.S. provisional serial No. 62/947,968 filed on 12/13/2019, incorporated herein in its entirety.

Claims

1.2-[3-[(3R) -3- [ [ 2-chloro-3- (trifluoromethyl) phenyl]Methyl- (2, 2-diphenylethyl) amino]Butoxy radical]Phenyl radical]Zinc salt of acetic acid (compound 1).

2. The zinc salt of claim 1, wherein said zinc salt is a 2:1 (compound 1: zinc) salt.

3. The zinc salt of claim 1 or 2, wherein the zinc salt is amorphous.

4. The zinc salt of any one of claims 1 to 3 having a mass loss due to decomposition of less than 1% as measured by thermogravimetric analysis.

5. Aluminum salt of compound 1.

6. The aluminum salt of claim 5, wherein the aluminum salt is a 3:1 (Compound 1: aluminum) salt.

7. The aluminium salt of claim 5 or 6, wherein the aluminium salt is amorphous.

8. The aluminium salt of any one of claims 5 to 7, which has a mass loss due to decomposition of less than 1% as measured by thermogravimetric analysis.

9. A pharmaceutical composition comprising a zinc or aluminium salt according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition comprises less than 1.5% by weight sodium.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is substantially free of the 1:1 sodium salt of compound 1.

12. The pharmaceutical composition of any one of claims 9-11, wherein the pharmaceutical composition is in a unit dosage form.

13. A method of treating cancer, the method comprising administering an effective amount of a zinc or aluminium salt of any one of claims 1 to 8 or a pharmaceutical composition of any one of claims 9 to 12.

14. The method of claim 13, wherein the cancer is breast cancer, colon cancer, renal cell carcinoma, lung cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, bladder cancer, head and neck cancer, glioblastoma, diffuse large B-cell lymphoma, leukemia, or melanoma.

15. The method of claim 14, wherein the cancer is a metastatic cancer.

16. The method of claim 15, wherein the effective amount comprises an amount effective to inhibit metastatic colonization of the cancer.

17. A method of producing a zinc salt of compound 1, the method comprising: combining compound 1 or a salt thereof and a zinc salt under conditions sufficient to produce the zinc salt of compound 1.

18. The method of claim 17, wherein the zinc salt of compound 1 is a 2:1 (compound 1: zinc) salt.

19. The method of claim 17 or 18, wherein compound 1 or the salt thereof is a 1:1 (compound 1: sodium) salt.

20. The method of any one of claims 17-19, wherein the zinc salt is zinc chloride.

21. The method of any one of claims 17-19, wherein the zinc salt is zinc acetate.

22. The method of any one of claims 17-21, wherein the method comprises dissolving compound 1 or a salt thereof and the zinc salt in a solvent to form a mixture.

23. The method of claim 22, wherein the method further comprises cycling the temperature of the mixture between ambient temperature and 40 ℃.

24. The method of claim 23, wherein the cycling is performed in a 4-hour period.

25. The method of claim 24, wherein the cycling is performed for 24 hours.

26. The zinc salt of claim 3, wherein the zinc salt has a particle size of about 1590 ± 10 cm as measured by fourier transform infrared spectroscopy (FTIR)^-1A peak with increased intensity relative to the free acid and a peak at about 1710 + -10 cm^-1A peak with reduced intensity relative to the free acid.